An alpha-1,2 fucosyltransferase mutant and 2'-fucosyllactose-producing escherichia coli

Abstract

The application discloses an alpha-1,2 fucosyltransferase mutant and an Escherichia coli for producing 2'-fucosyllactose, and belongs to the technical field of biotechnology. By means of bioinformatics and other methods, based on protein sequence alignment, a plurality of amino acid residues are selected as transformation targets by means of semi-rational selection of alanine scanning, a "small and precise" high-quality mutant library of alpha-1,2 fucosyltransferase is constructed, and a specific target is subjected to semi-rational transformation methods such as combined mutation, so that a high-activity alpha-1,2 fucosyltransferase mutant is obtained for synthesis of 2'-FL in the Escherichia coli, and a foundation is laid for development of food rich in 2'-fucosyllactose.

Description

Technical Field

[0001] This invention relates to an α-1,2-fucosyltransferase mutant and an Escherichia coli that produces 2′-fucosyllactose, belonging to the field of biotechnology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a class of novel functional sugars found in breast milk, second only to fat and lactose in abundance. They promote the growth of beneficial gut bacteria in newborns, strengthen the intestinal barrier, and play a crucial role in the development of the newborn's immune system. HMOs possess various core monosaccharide structural units. Based on the different spatial configurations, glycosylation sequences, and chain lengths of these monosaccharide structural units, HMOs can be classified into three categories: neutral fucoidan, acidic sialic acid lactose, and neutral non-fucosylated lactose. Among the many HMO oligosaccharide structures, approximately 50% are fucoidated, and 2′-fucosyllactose (2′-FL) is currently the most promising fucoidan. Due to its physiological functions such as inhibiting pathogenic bacterial infection, regulating intestinal flora, and enhancing immunity, it has been approved by several authoritative food safety risk assessment agencies both domestically and internationally as a nutritional additive in infant formula and special needs foods. Currently, the route for microbial metabolic synthesis of 2′-FL aligns with the green development requirements under the "dual carbon" goal, and thus gradually demonstrates enormous market potential in the food sector.

[0003] Fucosyltransferases (FucT) can transfer fucose from the donor substrate fucoguanosine diphosphate (GDP-L-fucose) to the acceptor substrate, synthesizing different types of human milk oligosaccharides. α-1,2-fucosyltransferases (FutC) are a key rate-limiting step in the synthesis of 2′-FL from the precursor GDP-L-fucose and the substrate lactose. α-1,2-fucosyltransferases derived from *Helicobacter pylori* (ATCC No. 26695) catalyze the transfer of the fucosyl group from the precursor GDP-L-fucose to the galactosyl group of the substrate lactose to synthesize 2′-FL. This enzyme can also catalyze the transfer of the fucosyl group to 2′-FL to generate the byproduct DFL. Therefore, screening for highly efficient α-1,2-fucosyltransferases is a key rate-limiting step affecting the efficiency of 2′-FL synthesis. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an α-1,2-fucosyltransferase mutant capable of high-yield production of 2′-fucosyllactose in Escherichia coli, and develops an efficient method for producing 2′-fucosyllactose based on this mutant, laying the foundation for the development of foods rich in 2′-fucosyllactose.

[0005] The first objective of this invention is to provide a recombinant *Escherichia coli* strain that produces 2′-fucosylated lactose, wherein the recombinant *Escherichia coli* strain heterologously expresses an α-1,2-fucosylation enzyme mutant, which is obtained by mutating the α-1,2-fucosylation enzyme with the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation is as follows:

[0006] The 14th position of asparagine was mutated to glutamine.

[0007] Alternatively, proline at position 116 could be mutated to glycine, and tyrosine at position 118 could be mutated to cysteine.

[0008] Or, the lysine at position 297 could be mutated to arginine.

[0009] Alternatively, glutamine at position 248 may be mutated to proline.

[0010] Furthermore, the recombinant Escherichia coli also includes the following modifications: the transcriptional repressor gene lacI, the undecyl glucose-1-phosphate transferase gene wcaJ, and the β-galactosidase gene lacZ were knocked out, and α-1,2-fucosyltransferase FutC and fucokinase Fkp were heterologously expressed.

[0011] Furthermore, the recombinant Escherichia coli uses Escherichia coli MG-26 as the starting strain. The genotype of this strain is MG1655△fliR::futC,△fuck::fkp,△lacI,△wcaJ,△lacZ,△flgA::futC,△flgG::futC.

[0012] Furthermore, using P tac The promoter (SEQ ID NO.2) initiates the expression of the gene encoding the α-1,2-fucosyltransferase mutant.

[0013] A second objective of this invention is to provide a method for producing 2′-fucosylated lactose, comprising the step of fermentation using the aforementioned recombinant Escherichia coli.

[0014] Furthermore, the fermentation process involves inoculating recombinant Escherichia coli into a seed culture medium to prepare a seed liquid, inoculating the seed liquid into a fermentation culture medium, and fermenting at 28-32℃ and 180-240 rpm.

[0015] Furthermore, the seed culture medium is LB medium.

[0016] Furthermore, the fermentation medium includes: glycerol 5-10 g / L, lactose 5-10 g / L, fucose 1-5 g / L, tryptone 10-15 g / L, yeast extract 20-30 g / L, dipotassium hydrogen phosphate 10-15 g / L, and potassium dihydrogen phosphate 1-5 g / L.

[0017] Furthermore, the inoculation amount is 1-10%, while the inoculation amount in this invention is 1-2%.

[0018] Furthermore, the fermentation time is 36-60 hours.

[0019] The method for de novo synthesis of 2′-FL using the fucosyltransferase mutant of the present invention comprises the following steps:

[0020] (1) Prediction of the FutC model structure

[0021] Based on the protein sequence alignment results of α-1,2-fucosyltransferases from different sources, the three-dimensional protein structure of FutC from Helicobacter pylori (ATCC No. 26695) was predicted using the structure prediction website AlphaFold2, and the conserved regions and loop of FutC were analyzed.

[0022] (2) Construction of FutC alanine scanning sites based on molecular docking

[0023] The flexible regions of enzymes are often related to their function and play an important role in catalytic reactions. Therefore, Autodock Vina software was first used to obtain complex conformations of FutC with different substrates (GDP-L-fucose and lactose) using a semi-flexible docking method. Subsequently, the loop near the substrate channel and the loop connecting the N-terminal and C-terminal domains were analyzed to determine the alanine scanning site. Finally, alanine mutants were constructed into the pMA09-futC plasmid through site-directed mutagenesis, and the constitutive promoter P was used. tac The gene was expressed, and the expression vector pMA09-MfutCA of the mutant was obtained through sequencing verification. Specifically, FutC was cloned into the pMA09 plasmid in a one-step process to obtain the recombinant plasmid pMA09-futC, which served as the control group for the FutC mutant.

[0024] (3) The effect of saturation mutations at key sites of the FutC flexible loop on 2′-FL synthesis

[0025] Based on the fermentation results of alanine scanning, degenerate primers (NNK or NDT) were designed to saturate-mutate amino acid sites near the substrate channel (N14, S43, P116, Y118, D120, R168, N248, I259, I279, K297), constructing a saturated mutant vector, which was then transformed into a 2′-FL synthesizing strain and fermented in 24-well plates. Liquid chromatography was used to detect the synthesis of 2′-FL.

[0026] A third objective of this invention is to provide the application of the aforementioned recombinant Escherichia coli in the preparation of food additives.

[0027] Furthermore, the food products include, but are not limited to, infant foods, such as those used in the preparation of formula milk powder.

[0028] The fourth object of this invention is to provide an α-1,2-fucosyltransferase mutant, which is obtained by mutating the α-1,2-fucosyltransferase with the amino acid sequence shown in SEQ ID NO.1, wherein the mutation is as follows:

[0029] The 14th position of asparagine was mutated to glutamine.

[0030] Alternatively, proline at position 116 could be mutated to glycine, and tyrosine at position 118 could be mutated to cysteine.

[0031] Or, the lysine at position 297 could be mutated to arginine.

[0032] Alternatively, glutamine at position 248 may be mutated to proline.

[0033] A fifth objective of this invention is to provide a nucleic acid molecule encoding the aforementioned α-1,2-fucosyltransferase mutant.

[0034] The sixth objective of this invention is to provide a recombinant plasmid carrying the aforementioned nucleic acid molecules.

[0035] A seventh objective of this invention is to provide host cells expressing the above-described α-1,2-fucosyltransferase mutant.

[0036] Furthermore, the host cell is a bacterium, fungus, plant cell, or animal cell.

[0037] An eighth object of the present invention is to provide the use of the above-mentioned α-1,2-fucosyltransferase mutant, nucleic acid molecule, recombinant plasmid or host cell in the preparation of 2′-fucosyllactose.

[0038] The beneficial effects of this invention are:

[0039] Currently, there are few reports on the catalytic mechanism of α-1,2-fucosyltransferases. Highly active α-1,2-fucosyltransferases are a key rate-limiting step in the synthesis of 2′-FL by catalyzing the transfer of GDP-L-fucose from the donor to the galactosyl group of the substrate lactose. To address this issue, this invention utilizes bioinformatics methods, based on protein sequence alignment and alanine scanning, to semi-rationally select multiple amino acid residues as modification targets. A small, high-quality mutant library of α-1,2-fucosyltransferases is constructed, and semi-rational modification methods such as combinatorial mutagenesis of specific targets are employed to obtain highly active α-1,2-fucosyltransferase mutants for the synthesis of 2′-FL in *E. coli*. Attached Figure Description

[0040] Figure 1 Protein sequence alignment of α-1,2-fucosyltransferases from different sources.

[0041] Figure 2 This is a schematic diagram of the three-dimensional structure of α-1,2-fucosyltransferase derived from Helicobacter pylori and its molecular docking with ligands GDP-L-fucose and lactose.

[0042] Figure 3 The results show the alanine scan of α-1,2-fucosyltransferase derived from Helicobacter pylori.

[0043] Figure 4 The effect of saturated mutants of α-1,2-fucosyltransferase from Helicobacter pylori on the synthesis of 2′-FL (* in the figure indicates that the mutated amino acid is a stop codon).

[0044] Figure 5 The results of shake-flask rescreening for the synthesis of 2′-FL using the α-1,2-fucosyltransferase mutant are shown. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0046] The method involved in this invention is as follows:

[0047] (1) Detection of 2′-FL:

[0048] To determine the yield of 2′-FL synthesized by the recombinant *E. coli* strain described in this invention and the consumption of the substrate lactose, a high-performance liquid chromatography (HPLC) system (Agilent Technologies 1260 series) was used to detect the synthesis of 2′-FL and the consumption of lactose in the fermentation broth of the engineered *E. coli* strain. Specifically, the concentration of 2′-FL in the fermentation supernatant was determined using an Aminex HPX-87H organic acid column. The HPLC detector was a differential detector, the mobile phase was 0.01N dilute H₂SO₄, the column detection temperature was set to 55℃, and the detection flow rate was set to 0.6 mL / min.

[0049] (2) Preparation method of Escherichia coli MG26 competent cells:

[0050] Glyceryl MG26 was streaked onto LB agar plates containing 50 μg / mL ampicillin and incubated at 37°C for 12 h. Single colonies were then picked and inoculated onto fresh LB agar containing 50 μg / mL ampicillin and incubated overnight at 37°C and 220 rpm. The overnight culture was then transferred at a 1% inoculum to 50 mL of supercompetent bacterial culture medium provided by the Beyotime kit and incubated at 37°C and approximately 220 rpm. The bacterial OD was then calculated. 600 When the pH reaches 0.6-0.7, incubate the bacterial suspension on ice for 15 min, then centrifuge at 4000×g for 5 min at 4℃ to collect the bacteria. Resuspend the bacterial pellet in 20 mL of pre-chilled supercompetent cell preparation reagent A, incubate on ice for 15 min, and centrifuge at 4000×g for 5 min at 4℃ to collect the bacteria. Then, resuspend the bacterial pellet in 2 mL of pre-chilled reagent B, incubate on ice for 15 min, and then aliquot to prepare E. coli MG26 competent cells for later use.

[0051] The present invention is as follows:

[0052] The genetically engineered strain of this invention was obtained by transforming the Escherichia coli MG26 (patent application number: CN202011240682.5) previously constructed in our laboratory into the FutC mutant vector pMA09-MfutC.

[0053] This invention first obtained the three-dimensional protein structure of FutC by submitting the amino acid sequence (SEQ ID NO.1) of FutC derived from Helicobacter pylori (ATCC No. 26695) to the AlphaFold2 structure prediction website. Subsequently, the structures of FutC, GDP-L-fucose, and lactose were optimized using Autodock Vina software. Semi-flexible docking was employed to obtain the docking conformations of the receptor FutC with the ligands GDP-L-fucose and lactose, respectively. Based on the molecular docking results, 40 amino acid sites on the loop of the flexible region near the binding pocket of FutC with the ligands were selected as alanine scanning sites for FutC. These sites were mutated to alanine and the constitutive promoter P was used. tac (SEQ ID NO.2) An alanine scanning mutant was expressed, and the expression vector pMA09-MfutC was constructed and transformed into MG26 cells. 24-well plate fermentation was then performed to investigate the effect of different alanine mutants on the loop on 2′-FL synthesis. Alanine scanning results showed that 10 positive mutants (N14A, S43A, P116A, Y118A, D120A, R168A, N248A, I259A, I279A, K297A) on the loop near the substrate channel promoted 2′-FL synthesis. These 10 amino acid sites were selected as saturation mutation sites for FutC. Degenerate primers NNK or NDT were designed, and the linearized vector pMA09-MFutC fragment was transformed into competent *E. coli* cells. After overnight culture, transformants were picked from the plates for 24-well plate fermentation. Finally, the 2′-FL synthesis was detected by liquid chromatography. This invention provides a simple method for constructing recombinant *E. coli* and has good application prospects.

[0054] Example 1

[0055] (1) Three-dimensional structure prediction of Helicobacter pylori α-1,2-fucosyltransferase

[0056] The amino acid sequence of FutC from *Helicobacter pylori* (ATCC No. 26695) was submitted to the AlphaFold2 structure prediction website (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / Alpha Fold2.ipynb) to predict the three-dimensional structure model of FutC. Simultaneously, based on the protein sequence alignment results of α-1,2-fucosyltransferases from different sources (see attached...),... Figure 1Four conserved motifs of α-1,2-fucosyltransferase were deduced: I8-20A (Motif 1), V165-D171 (Motif 2), V202-F209 (Motif 3), and I245-L258 (Motif 4). The structures of FutC, GDP-L-fucose, and lactose were then optimized using Autodock Vina software, yielding the receptor and ligand structure files futc.pdbqt, GDP-L-fucose.pdbqt, and lactose.pdbqt. Following the Autodock Vina software manual, a semi-flexible docking method was used (energy_range set to 4, num_modes set to 200, exhaustiveness set to 10) to obtain the docking conformations of the receptor FutC with the ligands GDP-L-fucose and lactose, respectively. Figure 2 It is speculated that S43-Q48 and K270-E283 are substrate channels for GDP-L-fucose and lactose binding of Helicobacter pylori α-1,2-fucosyltransferase.

[0057] (2) Selection and construction of alanine mutation site for Helicobacter pylori α-1,2-fucosyltransferase

[0058] The amino acids near these four conserved motifs, the loop near the substrate channel, and the 40 amino acid sites on the loop connecting the N-terminal and C-terminal domains were used as alanine scanning sites. Using the pMA09-futC plasmid as a template, the constitutive promoter P was employed. tac The expressed gene was amplified by PCR to obtain the alanine-directed mutant fragment of FutC, which was then purified and transformed into JM109 competent cells. Single colonies were picked for sequencing to verify the successful construction of the recombinant plasmid pMA09-MfutCA. These plasmids were then transformed into MG26 competent cells and fermented in 24-well plates to investigate the effect of different alanine mutants on 2′-FL synthesis. Alanine scanning results showed that 10 mutants (N14A, S43A, P116A, Y118A, D120A, R168A, N248A, I259A, I279A, K297A) on the loop near the substrate channel promoted 2′-FL synthesis. Figure 3 ).

[0059] (3) Construction of a saturated mutant library of key sites of the flexible loop of Helicobacter pylori α-1,2-fucosyltransferase

[0060] Based on the fermentation results of the alanine scan, further saturation mutations were performed on N14A, S43A, P116A, Y118A, D120A, R168A, N248A, I259A, I279A, and K297A to construct a saturation mutant library of FutC. Specifically, degenerate primers for these 10 sites were designed (Table 1). Using the pMA09-futC plasmid as a template, PCR amplification was performed to obtain linearized fragments of the saturation mutation sites. After recovery and purification, these fragments were transformed into JM109 competent cells. All recombinant plasmid mutant libraries were collected and transformed into MG26 cells. Integrative mutant recombinants were screened using 50 μg / mL ampicillin plates to obtain a recombinant E. coli mutant library.

[0061] (4) Screening and shake-flask rescreening of recombinant E. coli mutant libraries

[0062] Randomly selected *E. coli* mutant strains were inoculated as single colonies into LB medium in 24-well plates and cultured overnight at 30°C and 220 rpm. A 1% inoculum was then transferred to fermentation medium in 24-well plates containing: 5-10 g / L glycerol, 5-10 g / L lactose, 1-5 g / L fucose, 12 g / L tryptone, 24 g / L yeast extract, 12.54 g / L dipotassium hydrogen phosphate, and 2.31 g / L potassium dihydrogen phosphate, pH 6.5. Each well was filled to no more than 1 / 3 of its volume, and wells were capped with a breathable lid. The plates were incubated at 30°C and 700 rpm for 48 h. After fermentation, the plates were centrifuged at 8000 × g for 15 min at room temperature, and the supernatant was analyzed by HPLC to determine the concentration of 2′-FL. Among the *E. coli* mutant strains screened in 24-well plates, compared with the wild-type α-1,2-fucosyltransferase FutC, some mutants of N14, S43, P116Y118, D120, R168, N248, I259, I279, and K297 showed increased 2′-FL synthesis. Figure 4 Positive saturation mutants were selected from all saturation mutants for testing. Therefore, only the results of mutants with increased yield are shown in the figure; other mutants without increased yield are not shown.

[0063] Based on the above results, 12 mutant strains with superior yield (N14Q, S43V, S43D, P116GY118C, P116IY118I, P116VY118D, R168I, N248P, I259P, I279S, I279N, K297R) were selected for shake-flask re-screening. Single colonies were picked and inoculated into LB medium with a final concentration of ampicillin (50 μg / mL) and cultured for 8-10 h to serve as seed culture for shake-flask fermentation. The seed culture was then inoculated at a rate of 1% into a 250 mL Erlenmeyer flask containing 20 mL of fermentation medium (glycerol 5-10 g / L, lactose 5-10 g / L, fucose 1-5 g / L, tryptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L). Fermentation was carried out at 30 °C and 220 rpm for 48 h. The supernatant from the centrifuged fermentation at 48 h was analyzed by HPLC to determine the 2′-FL content. Eight plants that did not show a significant increase in yield were discarded after a second screening. Figure 5 The shake-flask yield of the control strain was 2.54 g / L. Among the four mutant strains obtained, MV4 had the highest yield, reaching 3.88 g / L, which was 152.8% of the original strain. The 2′-FL yields of the other mutant strains from MV1 to V3 were 3.68 g / L, 3.22 g / L, and 2.91 g / L, respectively. Further sequencing analysis of the α-1,2-fucosyltransferases in these four mutant strains revealed mutation sites of N14Q, P116GY118C, K297R, and N248P, respectively.

[0064] Table 1 Primers for saturation mutation sites of α-1,2-fucosyltransferase derived from Helicobacter pylori

[0065]

[0066]

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant *Escherichia coli* strain for producing 2'-fucosylated lactose, characterized in that, The recombinant *E. coli* heterologously expressed an α-1,2-fucosyltransferase mutant, which was obtained by mutating the α-1,2-fucosyltransferase with the amino acid sequence shown in SEQ ID NO.

1. The mutation is as follows: The 14th position of asparagine was mutated to glutamine. Alternatively, proline at position 116 could be mutated to glycine, and tyrosine at position 118 to cysteine. Or, the lysine at position 297 could be mutated to arginine. Alternatively, the asparagine at position 248 could be mutated to proline; The recombinant Escherichia coli used Escherichia coli MG-26 as the starting strain, with the genotype MG1655 △ fliR :: futC , △ fucK :: fkp , △ lacI , △ wcaJ , △ lacZ , △ flgA :: futC , △ flgG :: futC .

2. The recombinant Escherichia coli according to claim 1, characterized in that, Using P tac The promoter initiates the expression of the gene encoded by the α-1,2-fucosyltransferase mutant.

3. A method for producing 2'-fucosylated lactose, characterized in that, The step includes fermentation production using the recombinant Escherichia coli as described in claim 1 or 2.

4. The method according to claim 3, characterized in that, The fermentation process involves inoculating recombinant Escherichia coli into a seed culture medium to prepare a seed solution, then inoculating the seed solution into a fermentation culture medium and fermenting at 28-32℃ and 180-240 rpm.

5. The method according to claim 4, characterized in that, The fermentation medium comprises: 5-10 g / L glycerol, 5-10 g / L lactose, 1-5 g / L fucose, 10-15 g / L tryptone, 20-30 g / L yeast extract, 10-15 g / L dipotassium hydrogen phosphate, and 1-5 g / L potassium dihydrogen phosphate.

6. The use of the recombinant Escherichia coli according to claim 1 or 2 in the preparation of food additives.

7. The application according to claim 6, characterized in that, The food products mentioned include infant food.

8. An α-1,2-fucosyltransferase mutant, characterized in that, It was obtained by mutating the α-1,2-fucosyltransferase, whose amino acid sequence is shown in SEQ ID NO.

1. The mutation is as follows: The 14th position of asparagine was mutated to glutamine. Alternatively, proline at position 116 could be mutated to glycine, and tyrosine at position 118 to cysteine. Or, the lysine at position 297 could be mutated to arginine. Alternatively, the asparagine at position 248 may be mutated to proline.

9. The nucleic acid encoding the α-1,2-fucosyltransferase mutant of claim 8.

10. A recombinant plasmid carrying the nucleic acid of claim 9.

11. Host cells expressing the α-1,2-fucosyltransferase mutant of claim 8.

12. The use of the α-1,2-fucosyltransferase mutant of claim 8, the nucleic acid of claim 9, the recombinant plasmid of claim 10, or the host cell of claim 11 in the preparation of 2'-fucosyllactose.

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